Original Article Clinical Microbiology Ann Lab Med 2015;35:595-601 http://dx.doi.org/10.3343/alm.2015.35.6.595 ISSN 2234-3806 • eISSN 2234-3814
Distribution of β-Lactamase Genes Among Carbapenem-Resistant Klebsiella pneumoniae Strains Isolated From Patients in Turkey Meryem Iraz, M.D.1, Azer Özad Düzgün, M.S.2, Cemal Sandallı, Ph.D.3, Mehmet Ziya Doymaz, Ph.D.1, Yasemin Akkoyunlu, M.D.4, Ayşegül Saral, M.S.5, Anton Y. Peleg, Ph.D.6,7, Osman Birol Özgümüş, Ph.D.8, Fatih Şaban Beriş, Ph.D.3, Hakan Karaoğlu, Ph.D.9, and Ayşegül Çopur Çiçek, M.D.8 Department of Medical Microbiology1, Faculty of Medicine, Bezmialem Vakif University, Istanbul, Turkey; Department of Genetic and Bioengineering2, Faculty of Engineering and Natural Sciences, Gumushane University, Gumushane, Turkey; Department of Biology3, Faculty of Arts & Sciences, Recep Tayyip Erdoğan University, Rize, Turkey; Department of Infectious Diseases and Clinical Microbiology4, Faculty of Medicine, Bezmialem Vakif University, Istanbul, Turkey; Department of Biology5, Faculty of Arts & Sciences, ArtvinCoruh University, Artvin, Turkey; Department of Microbiology6, Monash University, Melbourne, Australia; Department of Infectious Diseases7, The Alfred Hospital, Melbourne, Australia; Department of Microbiology8, Faculty of Medicine, Recep Tayyip Erdoğan University, Rize, Turkey; Department of Aquaculture9, Faculty of Fisheries, Recep Tayyip Erdoğan University, Rize, Turkey
Background: The emergence of carbapenem-resistant Klebsiella pneumoniae poses a serious problem to antibiotic management. We investigated the β-lactamases in a group of carbapenem-resistant K. pneumoniae clinical isolates from Turkey.
Received: February 24, 2015 Revision received: April 24, 2015 Accepted: August 9, 2015
Methods: Thirty-seven strains of K. pneumoniae isolated from various clinical specimens were analyzed by antimicrobial susceptibility testing, PCR for the detection of β-lactamase genes, DNA sequencing, and repetitive extragenic palindronic (REP)- PCR analysis.
Corresponding author: Aysegul Çopur Çiçek Department of Medical Microbiology, Faculty of Medicine, Recep Tayyip Erdoğan University, 53020, Rize, Turkey Tel: +90-464-223-6126 Fax: +90-464-223-5376 E-mail: [email protected]
Results: All 37 isolates were resistant to ampicillin, ampicillin/sulbactam, piperacillin, piperacillin/tazobactam, ceftazidime, cefoperazone/sulbactam, cefepime, imipenem, and meropenem. The lowest resistance rates were observed for colistin (2.7%), tigecycline (11%), and amikacin (19%). According to PCR and sequencing results, 98% (36/37) of strains carried at least one carbapenemase gene, with 32 (86%) carrying OXA-48 and 7 (19%) carrying NDM-1. No other carbapenemase genes were identified. All strains carried a CTX-M-2-like β-lactamase, and some carried SHV- (97%), TEM- (9%), and CTX-M1-like (62%) β-lactamases. Sequence analysis of bla TEM genes identified a bla TEM-166 with an amino acid change at position 53 (Arg53Gly) from bla TEM-1b, the first report of a mutation in this region. REP-PCR analysis revealed that there were seven different clonal groups, and temporo-spatial links were identified within these groups. Conclusions: Combinations of β-lactamases were found in all strains, with the most common being OXA-48, SHV, TEM, and CTX-M-type (76% of strains). We have reported, for the first time, a high prevalence of the NDM-1 (19%) carbapenemase in carbapenem-resistant K. pneumoniae from Turkey. These enzymes often co-exist with other β-lactamases, such as TEM, SHV, and CTX-M β-lactamases. Key Words: bla TEM-166, Colistin, K. pneumoniae , NDM-1, OXA-48
INTRODUCTION Antibiotic resistance in gram-negative bacteria is a major problem http://dx.doi.org/10.3343/alm.2015.35.6.595
© The Korean Society for Laboratory Medicine This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/3.0) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
worldwide. Resistance to β-lactam antibiotics is often mediated by β-lactamases, including expanded-spectrum β-lactamases (ESBLs), plasmid-mediated AmpC β-lactamases, and carbapen-
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emases [1]. Clinical isolates of Klebsiella pneumoniae are notorious for harboring a wide range of β-lactamases and are naturally resistant to ampicillin and amoxycillin. β-Lactam antibiotics are often used in the treatment of infections caused by this bacterium [2]. The emergence and rapid spread of drug-resistant K. pneumoniae isolates has become a serious clinical challenge. β-Lactamases are grouped into four classes according to their amino acid sequence: class A, including KPC and TEM; class B, the metallo-β-lactamases (MBLs) such as VIM, IMP, and NDM1; class C, the AmpC β-lactamases; and class D, the oxacillinase (OXA)-type enzymes. All four classes of β-lactamases have been described in K. pneumoniae . Carbapenem resistance in K. pneumoniae has been reported in most countries and can be attributed to plasmid-mediated AmpC cephalosporinases associated with porin modifications [3, 4], reduced outer membrane permeability by porin loss in combination with the production of an ESBL, or production of β-lactamases capable of hydrolyzing carbapenems (carbapenemases) [5, 6]. In Turkey, OXA-48 carbapenemases have been identified for a decade [5], initially described in K. pneumoniae [7] and then in various other enterobacterial species [8-10]. Only recently, however, an imported case of NDM-1-producing K. pneumoniae has been reported in Turkey [11], but thus far, there have been no reports on locally acquired cases. In this study, we aimed to characterize the β-lactamases in carbapenem-resistant K. pneumoniae clinical isolates from Turkey. These data serve an important role in understanding and controlling the spread of carbapenem-resistant gram-negative pathogens [2].
zole, imipenem, meropenem, colistin, and tigecycline. Non-susceptibility to carbapenems (imipenem and meropenem) and colistin during susceptibility testing was confirmed by E-test (bioMerieux) in accordance with the CLSI guidelines [13].
2. Detection of β-lactamase genes
K. pneumoniae strains were screened for β-lactamase-encoding genes by PCR. The primers used to amplify the bla SHV, bla TEM, bla CTX-M1, bla CTX-M2, bla GES, bla VEB, bla PER-2, bla NDM-1, bla IMP-like, bla VIMlike, bla KPC and bla OXA-48 genes are listed in Table 1. A single reaction mixture contained 5 μL genomic DNA, 20 pM of each primer, 10 μL reaction buffer, 3 μL 25 mM MgCl2, 200 μM dNTPs, and 1.5 U Go Taq Flexi Polymerase (Promega, Madison, WI, USA), in a final volume of 50 μL. PCR amplification was performed by using standard conditions with varying annealing temperatures (Table 1). All PCR results were analyzed on 1% Table 1. Primers used for amplication and sequencing in this study
Primer GES VEB
863
56
542
55
927
50
488
56
780
58
813
54
260
50
341
50
860
49
843
55
743
57
796
52
[15]
F: ATTTCCCGATGCAAAGCGT R: TTATTCCGGAAGTCCCTGT
PER-2
F: ATGAATGTCATCACAAAATG R: TCAATCCGGACTCACT
IMP
F: CATGGTTTGGTGGTTCTTGT R: ATAATTTGGCGGACTTTGGC
METHODS
F: ATTGGTCTATTTGACCGCGTC R: TGCTACTCAACGACTGAGCG
NDM
1. Bacterial strains and antibiotic susceptibilities
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F: ATGCGCTTCATTCACGCAC
Amplicon Tm Reference size (bp) (°C)
R: CTATTTGTCCGTGCTCAGGA
VIM
The study was performed at the Bezmialem Vakif University Hospital in Turkey between July 2012 and March 2013. During the study period, all non-duplicate clinical isolates of K. pneumoniae obtained from inpatients that exhibited a reduced sensitivity to carbapenems were characterized. Clinical isolates were identified by using standard microbiological procedures and VITEK-2 (bioMerieux, Marcy-l’Etoile, France). The identification of all isolates was confirmed by 16S rDNA sequencing [12]. Susceptibility testing was performed by using the following antibiotics: ampicillin, ampicillin/sulbactam, piperacillin, piperacillin/ tazobactam, cefuroxime, ceftazidime, cefoperazone/sulbactam, cefepime, amikacin, gentamicin, netilmicin, tobramycin, ciprofloxacin, levofloxacin, tetracycline, trimethoprim/sulfamethoxa-
5´-3´ Sequence
F: TGGAATTGCCCAATATTATGC R: TCAGCGCAGCTTGTCGGCCATGC
CTX-M1 group CTX-M2 group TEM
F: GCGTGATACCACTTCACCTC
[16]
R: TGAAGTAAGTGACCAGAATC F: TGATACCACCACGCCGCTC R: TATTGCATCAGAAACCGTGGG F: AGTATTCAACATTTYCGTGT R: TAATCAGTGAGGCACCTATCTC
SHV
F: ATGCGTTATATTCGCCTGTG R: TTAGCGTTGCCAGTGCTC
OXA-48
A: TTGGTGGCATCGATTATCGG
[7]
B: GAGCACTTCTTTTGTGATGGC KPC
A: CGTTCTTGTCTCTCATGGCC B: CCTCGCTGTGCTTGTCATCC
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Iraz M, et al. β-Lactamase genes in carbapenem-resistant K. pneumoniae
agarose containing 0.5 μg/mL ethidium bromide and subsequently visualized under UV light. The PCR products for NDM, OXA-48, and TEM β-lactamases were cloned into the pGEM-T Easy vector (Promega) and then sequenced by Macrogen (Amsterdam, The Netherlands). Sequencing results were analyzed by using the alignment search tool, BLAST (http://www.ncbi. nlm.nih.gov/BLAST) [14], and the multiple sequence aligment program CLUSTALW2 (http://www.ebi.ac.uk/Tools/msa/clustalw2/). The remaining genes were evaluated according to their molecular size and PCR results of control groups which were defined as β-lactamase gene carrier bacteria in the earlier studies [7, 15, 16].
3. Repetitive Extragenic Palindronic (REP)-PCR analysis REP-PCR was used in the genotyping of K. Pneumoniae isolates. The primer pair REP 1 (5´-IIIGCGCCGICATCAGGC-3´) and REP 2 (5´-ACGTCTTATCAGGCCTAC-3´) was used [17], and the amplification reaction was performed as previously described [15]. The amplified DNA fragments were separated by electrophoresis on a 1.5% agarose gel with a 1-kb DNA ladder (Sigma Chemicals, Ontario, Canada) as the size marker. The amplified DNA bands were visualized under UV light following ethidium bromide staining, and the banding patterns of each strain were captured by using an UVP bioimaging system (UVP, Upland, CA, USA). The REP-PCR fingerprints were analyzed by using the Phoretix gel analysis package (Nonlinear USA, Inc., Durham, NC, USA).
RESULTS A total of 37 K. pneumoniae isolates were identified during the study period and investigated. All isolates were from hospitalacquired infections, with the majority from patients in the intensive care unit (n = 23), followed by patients on surgical (n = 7) and medical wards (n = 7). Of the 37 isolates, 16 (43%) were
associated with bloodstream infections. The remaining isolates came from wounds (n = 7, 19%), sputum (n = 6, 16%), tracheal aspirates (n = 6, 16%), cerebrospinal fluid (n = 1, 3%), and a catheter sample (n = 1, from anesthesia unit). According to the antibiotic susceptibility profiles, all 37 isolates were resistant to ampicillin, ampicillin/sulbactam, piperacillin, piperacillin/tazobactam, ceftazidime, cefoperazone/sulbactam, cefepime, imipenem, and meropenem. The lowest levels of resistance were observed for colistin (2.7%) and tigecycline (11%), followed by amikacin (19%) and trimethoprim/sulphamethoxazole (21.6%). Of the 37 K. pneumoniae isolates, 98% carried a carbapenemase gene. More specifically, we detected OXA-48 in 32 isolates (86%) and NDM-1 in seven isolates (19%). No other carbapenemases were identified within this group. We also observed a high prevalence of ESBLs, with all strains carrying a CTX-M-2 group β-lactamase and 23 isolates (62%) carrying CTX-M-1. SHV-1-like and TEM-1-like β-lactamase were also common (97% and 92% of strains, respectively). One of the bla TEM genes was identified as bla TEM-166, a variant of bla TEM-1b that has one amino acid change at position 53 (Arg53Gly). The combination of β-lactamases relative to the site of isolation and susceptibility test results are shown in Table 2. Importantly, all NDM-1-producing strains were isolated from blood. Furthermore, all blood isolates carried at least one carbapenemase (OXA-48 or NDM-1), and two (13%) carried both OXA-48 and NDM-1. The most common combination of β-lactamases in a given strain was OXA-48, SHV-1-like, TEM-1-like and CTX-Mtype β-lactamase, with 76% of strains observed with this combination. All 37 K. pneumoniae strains were typed by REP-PCR (Fig. 1). We observed seven predominant genotypes; the majority of strains (46%) clustered into genotype 6, while 19% and 16% of the isolates clustered in genotype 3 and 1, respectively. Of the 17 genotype 6 isolates, 11 were isolated from the anesthesia
Fig. 1. Repetitive extragenic palindronic (REP)-PCR profiles of 37 Klebsiella pneumoniae isolates. Seven different genotypes were observed, and most isolates clustered into genotypes 1, 3, and 6. http://dx.doi.org/10.3343/alm.2015.35.6.595
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Table 2. Epidemiological properties of β-lactamase-producing Klebsiella pneumoniae isolates
Isolate
Specimen
Units
Dates
Treatment
Outcome
Susceptible antibiotics
bla NDM-1 bla OXA-48 bla CTX-M1 bla CTX-M2 bla TEM
bla SHV
1
Blood
AR
Feb. 2013 SCF, CIP, TIG
Exitus
AK, TIG, CST, SXT
-
-
+
+
+
+
2
Blood
Gas
Feb. 2013 NT
Exitus
AK, CN, TIG, CST, SXT
-
+
+
+
-
+
3
Blood
Neu
Feb. 2013 TZP, SXT
Other
CN, TIG, CST, SXT
+
+
+
+
+
+
4
Blood
Neu
Feb. 2013 SXT, SCF
Other
TIG, CST, SXT
+
-
+
+
+
+
5
Blood
TS
Jan. 2013 NT
Other
AK, TIG, CST
-
+
+
+
+
+
6
Sputum
TS
Jan. 2013 NT
Other
AK, CST
-
+
+
+
+
+
7
Wound
RO
Jan. 2013 NT
Other
AK, CN, TIG, CST, SXT
-
+
+
+
-
+
8
Sputum
TS
Jan. 2013 NT
Other
AK, TIG, CST, SXT
-
+
+
+
+
+
9
Blood
IM
Jan. 2013 SCF, TIG
Other
AK, TIG, CST, SXT
-
+
+
+
+
+
10
Sputum
RICU
Jan. 2013 NT
Other
AK, CN, NET, TOB, TIG, CST, SXT
-
+
-
+
+
+
11
Sputum
RICU
Jan. 2013 SCF, TIG
Exitus
AK, CN, NET, TOB, TIG, CST, SXT
-
+
-
+
+
+
12
Wound
TS
Jan. 2013 CST, TIG
Other
AK, CST
-
+
+
+
+
+
13
Blood
PS
Jan. 2013 NT
Other
TIG, CST
+
+
+
+
+
+
14
Blood
AR
Jan. 2013 SCF, CST
Exitus
AK, CN, NET, TOB, TIG, CST, SXT
+
-
+
+
+
+
15
Tracheal aspirate
AR
Dec. 2012 SCF, TIG
Exitus
AK, TIG, CST, SXT
-
+
+
+
+
+
16
Blood
AR
Dec. 2012 TIG, SXT, CST
Exitus
AK, TIG, CST, SXT
-
+
+
+
+
+
17
Blood
Uro
Dec. 2012 NT
Other
AK, CN, TIG, CST, SXT
+
-
+
+
+
+
18
Wound
GS
Dec. 2012 MEM, TIG
Other
AK, CIP, LEV, TET, TIG, CST, SXT
-
+
+
+
-
+
19
Tracheal aspirate
AR
Dec. 2012 TIG, SUL
Treated
CN, TIG, CST, SXT
-
+
+
+
+
+
20
Blood
AR
Dec. 2012 CST, TIG
Exitus
AK, TIG, CST, SXT
-
+
+
+
+
+
21
Catheter
AR
Dec. 2012 TIG, CST
Exitus
AK, TIG, CST, SXT
-
+
+
+
+
+
22
CSF
AR
Nov. 2012 SXT, MEM, CIP
Exitus
AK, TIG, CST, SXT
-
+
+
+
+
+
23
Sputum
RICU
Feb. 2013 SCF, TIG
Exitus
AK, CN, TIG, CST, SXT
-
+
-
+
+
+
24
Blood
RICU
Oct. 2012 TIG, SUL
Exitus
TIG, SXT
-
+
-
+
+
+
25
Blood
AR
Oct. 2012 TIG, SUL
Exitus
TIG, CST, SXT
+
-
-
+
+
+
26
Wound
Gas
Nov. 2012 NT
Other
AK, TET, TIG, CST, SXT
-
+
-
+
+
-
27
Wound
AR
Jul. 2012 MEM, CIP, TIG, AK
Other
CN, CST
-
+
+
+
+
+
28
Blood
AR
Oct. 2012 SCF, CIP
Other
AK, TIG, CST, SXT
-
+
-
+
+
+
29
Wound
AR
Oct. 2012 CST, SUL, TIG, AK
Exitus
AK, TIG, CST, SXT
-
+
-
+
+
+
30
Tracheal aspirate
AR
Nov. 2012 TIG, RIF
Other
AK, TIG, CST, SXT
-
+
-
+
+
+
31
Tracheal aspirate
AR
Nov. 2012 TIG, SUL, CN, MEM, CST
Exitus
AK, TIG, CST, SXT
-
+
-
+
+
+
32
Tracheal aspirate
AR
Nov. 2012 NT
Exitus
AK, TIG, CST, SXT
-
+
-
+
+
+
33
Blood
AR
Aug. 2012 CST, CSF, TIG
Other
AK, CN, CST
-
+
-
+
+
+
34
Wound
Uro
Jul. 2012 NT
Other
AK, CN, TIG, CST
-
+
+
+
+
+
35
Sputum
AR
Nov. 2012 CST, SUL, TIG
Exitus
AK, TIG, CST, SXT
-
+
+
+
+
+
36
Blood
AR
Sep. 2012 CST, TIG
Other
AK, TIG, CST
-
+
-
+
+
+
37
Tracheal aspirate
AR
Nov. 2012 NT
Other
AK, TIG, CST, SXT
-
+
-
+
+
+
Abbreviations: AR, anesthesiology and reanimation; Gas, gastroenterology; Neu, neurology; TS, thoracic surgery; RO, radiation oncology; CP, chest diseases polyclinic; IM, internal medicine; RICU, respiratory intensive care unit; PS, plastic surgery; Uro, urology; GS, general surgery; TZP, piperacillin/tazobactam; SCF, cefaperazone/sulbactam; AK, amikacin; CN, gentamicin; NET, netilmicin; TOB, tobramycin; CIP, ciprofloxacin; LEV, levofloxacin; TET, tetracycline; TIG, tigecycline; CST, colistin; SXT, trimethoprim/sulfamethoxazole; MEM, meropenem; SUL, sulbactam; RIF, rifampicin; CSF, cerebrospinal fluid; NT, Not treated; Other, the patient was transferred into a different service.
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and reanimation intensive care unit (ICU), and three were isolated from the thoracic surgery unit. Of the seven genotype 3 isolates, two were isolated from patients who were in the neurology ward at the same time, and two were isolated from the anesthesia and ICU. The remaining isolates were distributed among other wards in the hospital over different times during the 9-month study period (Table 2).
DISCUSSION K. pneumoniae is an opportunistic pathogen that causes community- and hospital-acquired infections, such as bloodstream and urinary tract infections, and pneumonia [18]. Currently, carbapenems are the most potent antimicrobial agents used in the treatment of serious infections caused by multidrug-resistant gram-negative bacteria. However, carbapenem resistance in Enterobacteriaceae is emerging in Turkey [8, 9] and in other parts of the world [5]. Here, we described the distribution of major β-lactamases in a contemporary group of extensively drug-resistant K. pneumoniae isolates from Turkey. We observed that 19% of isolates carried the NDM-1 carbapenemase, and that all of these isolates came from bloodstream infections and from patients with no recent travel history, suggesting local acquisition. OXA-48 carbapenemases were highly prevalent, and were occasionally found co-existing with NDM-1. Compounding these data was that over 2.7% of isolates were resistant to colistin, a finding that is novel for Turkey and one with serious therapeutic implications. The majority of isolates came from patients in the ICU. BLs have been reported worldwide, and their genes are often located on plasmids and integrons [19]. NDM is a novel MBL enzyme, and its variants are the latest carbapenemases to be recognized and reported worldwide. NDM-1 was first identified in a carbapenem-resistant K. pneumoniae strain from the urine sample of a Swedish patient of Indian origin who traveled to New Delhi [20]. Since then, more than 40 countries have reported NDM-producing isolates [21, 22]. NDM enzymes have predominately been identified in Enterobacteriaceae , but have also been described in non-fermenters and in members of the Vibrionaceae family [23]. Occasional community-acquired NDM-1-positive isolates have been reported [24], however, the overwhelming majority of isolates have a nosocomial origin. In our study, all isolates were from nosocomial infections and all were from seriously ill patients. All of the NDM-1-positive isolates (n = 7) were recovered from blood samples, and six of these were from patients in the ICU. All pahttp://dx.doi.org/10.3343/alm.2015.35.6.595
tients were uninfected at admission, and the organisms were isolated from patients 48 hr or longer after admission. In Turkey, NDM-1 was first reported in 2011 in a K. pneumoniae isolate from a leukemia patient originally from Iraq [19]. Later, another team investigated NDM-1 in various carbapenemase-resistant gram-negative bacilli, but with negative results [25]. To our knowledge, the current study is the largest report demonstrating the presence of NDM-1 in K. pneumoniae in Turkey. Our results indicate that seven out of 37 (19%) carbapenem-resistant K. pneumonia isolates were positive for NDM-1, an alarming result as efforts to identify NDM-1-producing isolates in 2011 showed negative results. Another β-lactamase type known as OXA-48 was initially identified in Turkey from a K. pneumoniae isolate from Istanbul, and thereafter, outbreaks of OXA-48-producing K. pneumoniae have been described in Turkey [8-10]. To date, 11 variants of OXA-48 have been described [26]. Recently, the molecular characterization of carbapenem-resistant K. pneumoniae in a tertiary university hospital in Turkey was performed on 94 isolates. OXA-48 and NDM-1 were produced in 91.5% and 4.3% of isolates, respectively [27]. In our study, OXA-48 and NDM-1 were observed in 86% and 19% of isolates, respectively. This study reveals that the NDM-1 resistance gene is increasing in prevalence in Turkey. This increase poses a significant threat to Turkish infection control efforts. TEM or SHV β-lactamase derivatives have been the most prevalent ESBLs in nosocomial pathogens since the 1980s. However, starting from 1995, CTX-M-type ESBLs have emerged worldwide, including Europe, Asia, South America, and Canada [13, 28]. We demonstrated that these CTX-M-type ESBLs are also highly prevalent in multidrug-resistant K. pneumoniae in Turkey. In a multicenter study conducted in Turkish hospitals, PCR analysis was used for ESBL typing, and CTX-M was the most prevalent enzyme type among blood isolates (71%) of Escherichia coli and K. pneumonia , followed by TEM (49%) and SHV (47%) type enzymes [29]. The CTX-M family can be classified into five major groups: CTX-M-1, CTX-M-2, CTX-M-8, CTX-M-9, and CTX-M-25. CTXM-15, which belongs to the CTX-M-1 group, is the most widespread of these enzymes around the world [30]. Ours is the first report of CTX-M-1 and CTX-M-2 in ESBL-producing carbapenem-resistant K. pneumoniae strains from Turkey. CTX-M-2 was the most prevalent β-lactamase, found in 100% of isolates, followed by SHV (97%), TEM (92%), and CTX-M-1 (62%). The continued monitoring of antimicrobial resistance to β-lactams in each region of Turkey is urgently required to assist with the de-
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velopment of control strategies for hospital infections. In conclusion, our study of carbapenem-resistant, K. pneumoniae isolates identified the largest number of NDM-1-producing strains reported from Turkey, and revealed that these strains were highly resistant to a range of clinically important antibiotics. Furthermore, these isolates often carried the OXA-48 carbapenemase, and were associated with serious infections of the bloodstream. All strains also carried multiple ESBLs, with bla CTX-M-2 being the most common. The recent emergence of NDM-1-producing K. pneumoniae on the background of the highly prevalent OXA-48 and CTX-M type ESBLs is alarming for our country.
Acknowledgments This work was supported by Recep Tayyip Erdoğan University Research Fund Grants BAP-2013.102.03.12 and BAP-2013. 102.03.13. A.Y.P was supported by an Australian National Health and Medical Research Council Career Development Fellowship (APP1047916).
Authors’ Disclosures of Potential Conflicts of Interest No potential conflicts of interest relevant to this article were reported.
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7. Poirel L, Héritier C, Tolün V, Nordmann P. Emergence of oxacillinasemediated resistance to imipenem in Klebsiella pneumoniae . Antimicrob Agents Chemother 2004;48:15-22. 8. Aktaş Z, Kayacan CB, Schneider I, Can B, Midilli K, Bauernfeind A. Carbapenem-hydrolyzing oxacillinase, OXA-48, persists in Klebsiella pneumonia in Istanbul, Turkey. Chemotherapy 2008;54:101-6. 9. Carrër A, Poirel L, Eraksoy H, Cagatay AA, Badur S, Nordmann P. Spread of OXA-48-positive carbapenem-resistant Klebsiella pneumonia isolates in Istanbul, Turkey. Antimicrob Agents Chemother 2008;52: 2950-4. 10. Gülmez D, Woodford N, Palepou MF, Mushtaq S, Metan G, Yakupogullari Y, et al. Carbapenem-resistant Escherichia coli and Klebsiella pneumonia isolates from Turkey with OXA-48-like carbapenemases and outer membrane protein loss. Int J Antimicrob Agents 2008;31:523-6. 11. Poirel L, Ozdamar M, Ocampo-Sosa AA, Türkoğlu S, Ozer UG, Nordmann P. NDM-1- producing Klebsiella pneumoniae now in Turkey. Antimicrob Agents Chemother 2012;56:2784-5. 12. Copur Cicek A, Ozad Duzgun A, Saral A, Sandalli C. Determination of a novel integron-located variant (bla OXA-320) of Class D β-lactamase in Proteus mirabilis . J Basic Microbiol 2014;54:1030-5. 13. Clinical and Laboratory Standards Institute. Performance standards for antimicrobial susceptibility testing. M100-S23. Wayne, PA: Clinical and Laboratory Standards Institute, 2013. 14. Altschul SF, Madden TL, Schäffer AA, Zhang J, Zhang Z, Miller W, et al. Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res 1997;25:3389-402. 15. Copur Cicek A, Saral A, Iraz M, Ceylan A, Duzgun AO, Peleg AY, et al. OXA- and GES-type β-lactamases predominate in extensively drug-resistant Acinetobacter baumannii isolates from a Turkish University Hospital. Clin Microbiol Infect 2014;20:410-5. 16. Copur Cicek A, Saral A, Ozad Duzgun A, Yasar E, Cizmeci Z, Ozlem Balci P, et al. Nationwidestudy of Escherichia coli producing extended-spectrum β-lactamases TEM, SHV and CTX-M in Turkey. J Antibiot 2013; 66:647-50. 17. Andriamanantena TS, Ratsima E, Rakotonirina HC, Randrianirina F, Ramparany L, Carod JF, et al. Dissemination of multidrug resistant Acinetobacter baumannii in various hospitals of Antananarivo Madagascar. Ann Clin Microbiol Antimicrob 2010;9:17. 18. Podschun R and Ullman U. Klebsiella spp. as nosocomial pathogens: epidemiology, taxonomy, typing methods, and pathogenicity factors. Clin Microbiol Rev 1998;11:589-603. 19. Poirel L, Pitout JD, Nordmann P. Carbapenemases: molecular diversity and clinical consequences. Future Microbiol 2007;2:501-12. 20. Yong D, Toleman MA, Giske CG, Cho HS, Sundman K, Lee K, et al. Characterization of a new metallo-beta-lactamase gene, bla(NDM-1), and a novel erythromycin esterase gene carried on a unique genetic structure in Klebsiella pneumoniae sequence type 14 from India. Antimicrob Agents Chemother 2009;53:5046-54. 21. Johnson AP and Woodford N. Global spread of antibiotic resistance: the example of New Delhi metallo-β-lactamase (NDM)-mediated carbapenem resistance. J Med Microbiol 2013;62:499-513. 22. Seija V, Medina Presentado JC, Bado I, Papa Ezdra R, Batista N, Gutierrez C, et al. Sepsis caused by New Delhi metallo-β-lactamase (blaNDM-1) and qnrD-producing Morganella morganii, treated successfully with fosfomycin and meropenem: case report and literature review. Int J Infect Dis 2015;30:20-6. 23. Nordmann P, Poirel L, Walsh TR, Livermore DM. The emerging NDM carbapenemases. Trends Microbiol 2011;19:588-95. 24. Kim SY, Rhee JY, Shin SY, Ko KS. Characteristics of community-onset NDM-1-producing Klebsiella pneumonia isolates. J Med Microbiol
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2014;63:86-9. 25. Rasheed JK, Kitchel B, Zhu W, Anderson KF, Clark NC, Ferraro MJ, et al. New Delhi metallo-β-lactamase-producing Enterobacteriaceae, United States. Emerg Infect Dis 2013;19:870-8. 26. Evans B and Amyes SG. OXA β-lactamases. Clin Microbiol Rev 2014; 27:241-63. 27. Alp E, Perçin D, Colakoğlu S, Durmaz S, Kürkcü CA, Ekincioğlu P, et al. Molecular characterization of carbapenem-resistant Klebsiella pneumoniae in a tertiary university hospital in Turkey. J Hosp Infect 2013;84: 178-80. 28. Pitout JD, Nordmann P, Laupland KB, Poirel L. Emergence of Entero-
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bacteriaceae producing extended-spectrum β-lactamases (ESBLs) in the community. J Antimicrob Chemother 2005;56: 52-9. 29. Gür D, Gülay Z, Akan OA, Aktaş Z, Kayacan CB, Cakici O, et al. Resistance to newer beta-lactams and related ESBL types in gram-negative nosocomial isolates in Turkish hospitals: results of the multicentre HITIT study. Mikrobiyol Bul 2008;42:537-44. 30. Onnberg A, Mölling P, Zimmermann J, Söderquist B. Molecular and phenotypic characterization of Escherichia coli and Klebsiella pneumoniae producing extended-spectrum β-lactamases with focus on CTX-M in a low-endemic area in Sweden. APMIS 2011;119:287-95.
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Distribution of β-Lactamase Genes Among Carbapenem-Resistant Klebsiella pneumoniae Strains Isolated From Patients in Turkey Meryem Iraz, M.D.1, Azer Özad Düzgün, M.S.2, Cemal Sandallı, Ph.D.3, Mehmet Ziya Doymaz, Ph.D.1, Yasemin Akkoyunlu, M.D.4, Ayşegül Saral, M.S.5, Anton Y. Peleg, Ph.D.6,7, Osman Birol Özgümüş, Ph.D.8, Fatih Şaban Beriş, Ph.D.3, Hakan Karaoğlu, Ph.D.9, and Ayşegül Çopur Çiçek, M.D.8 Department of Medical Microbiology1, Faculty of Medicine, Bezmialem Vakif University, Istanbul, Turkey; Department of Genetic and Bioengineering2, Faculty of Engineering and Natural Sciences, Gumushane University, Gumushane, Turkey; Department of Biology3, Faculty of Arts & Sciences, Recep Tayyip Erdoğan University, Rize, Turkey; Department of Infectious Diseases and Clinical Microbiology4, Faculty of Medicine, Bezmialem Vakif University, Istanbul, Turkey; Department of Biology5, Faculty of Arts & Sciences, ArtvinCoruh University, Artvin, Turkey; Department of Microbiology6, Monash University, Melbourne, Australia; Department of Infectious Diseases7, The Alfred Hospital, Melbourne, Australia; Department of Microbiology8, Faculty of Medicine, Recep Tayyip Erdoğan University, Rize, Turkey; Department of Aquaculture9, Faculty of Fisheries, Recep Tayyip Erdoğan University, Rize, Turkey
Background: The emergence of carbapenem-resistant Klebsiella pneumoniae poses a serious problem to antibiotic management. We investigated the β-lactamases in a group of carbapenem-resistant K. pneumoniae clinical isolates from Turkey.
Received: February 24, 2015 Revision received: April 24, 2015 Accepted: August 9, 2015
Methods: Thirty-seven strains of K. pneumoniae isolated from various clinical specimens were analyzed by antimicrobial susceptibility testing, PCR for the detection of β-lactamase genes, DNA sequencing, and repetitive extragenic palindronic (REP)- PCR analysis.
Corresponding author: Aysegul Çopur Çiçek Department of Medical Microbiology, Faculty of Medicine, Recep Tayyip Erdoğan University, 53020, Rize, Turkey Tel: +90-464-223-6126 Fax: +90-464-223-5376 E-mail: [email protected]
Results: All 37 isolates were resistant to ampicillin, ampicillin/sulbactam, piperacillin, piperacillin/tazobactam, ceftazidime, cefoperazone/sulbactam, cefepime, imipenem, and meropenem. The lowest resistance rates were observed for colistin (2.7%), tigecycline (11%), and amikacin (19%). According to PCR and sequencing results, 98% (36/37) of strains carried at least one carbapenemase gene, with 32 (86%) carrying OXA-48 and 7 (19%) carrying NDM-1. No other carbapenemase genes were identified. All strains carried a CTX-M-2-like β-lactamase, and some carried SHV- (97%), TEM- (9%), and CTX-M1-like (62%) β-lactamases. Sequence analysis of bla TEM genes identified a bla TEM-166 with an amino acid change at position 53 (Arg53Gly) from bla TEM-1b, the first report of a mutation in this region. REP-PCR analysis revealed that there were seven different clonal groups, and temporo-spatial links were identified within these groups. Conclusions: Combinations of β-lactamases were found in all strains, with the most common being OXA-48, SHV, TEM, and CTX-M-type (76% of strains). We have reported, for the first time, a high prevalence of the NDM-1 (19%) carbapenemase in carbapenem-resistant K. pneumoniae from Turkey. These enzymes often co-exist with other β-lactamases, such as TEM, SHV, and CTX-M β-lactamases. Key Words: bla TEM-166, Colistin, K. pneumoniae , NDM-1, OXA-48
INTRODUCTION Antibiotic resistance in gram-negative bacteria is a major problem http://dx.doi.org/10.3343/alm.2015.35.6.595
© The Korean Society for Laboratory Medicine This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/3.0) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
worldwide. Resistance to β-lactam antibiotics is often mediated by β-lactamases, including expanded-spectrum β-lactamases (ESBLs), plasmid-mediated AmpC β-lactamases, and carbapen-
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emases [1]. Clinical isolates of Klebsiella pneumoniae are notorious for harboring a wide range of β-lactamases and are naturally resistant to ampicillin and amoxycillin. β-Lactam antibiotics are often used in the treatment of infections caused by this bacterium [2]. The emergence and rapid spread of drug-resistant K. pneumoniae isolates has become a serious clinical challenge. β-Lactamases are grouped into four classes according to their amino acid sequence: class A, including KPC and TEM; class B, the metallo-β-lactamases (MBLs) such as VIM, IMP, and NDM1; class C, the AmpC β-lactamases; and class D, the oxacillinase (OXA)-type enzymes. All four classes of β-lactamases have been described in K. pneumoniae . Carbapenem resistance in K. pneumoniae has been reported in most countries and can be attributed to plasmid-mediated AmpC cephalosporinases associated with porin modifications [3, 4], reduced outer membrane permeability by porin loss in combination with the production of an ESBL, or production of β-lactamases capable of hydrolyzing carbapenems (carbapenemases) [5, 6]. In Turkey, OXA-48 carbapenemases have been identified for a decade [5], initially described in K. pneumoniae [7] and then in various other enterobacterial species [8-10]. Only recently, however, an imported case of NDM-1-producing K. pneumoniae has been reported in Turkey [11], but thus far, there have been no reports on locally acquired cases. In this study, we aimed to characterize the β-lactamases in carbapenem-resistant K. pneumoniae clinical isolates from Turkey. These data serve an important role in understanding and controlling the spread of carbapenem-resistant gram-negative pathogens [2].
zole, imipenem, meropenem, colistin, and tigecycline. Non-susceptibility to carbapenems (imipenem and meropenem) and colistin during susceptibility testing was confirmed by E-test (bioMerieux) in accordance with the CLSI guidelines [13].
2. Detection of β-lactamase genes
K. pneumoniae strains were screened for β-lactamase-encoding genes by PCR. The primers used to amplify the bla SHV, bla TEM, bla CTX-M1, bla CTX-M2, bla GES, bla VEB, bla PER-2, bla NDM-1, bla IMP-like, bla VIMlike, bla KPC and bla OXA-48 genes are listed in Table 1. A single reaction mixture contained 5 μL genomic DNA, 20 pM of each primer, 10 μL reaction buffer, 3 μL 25 mM MgCl2, 200 μM dNTPs, and 1.5 U Go Taq Flexi Polymerase (Promega, Madison, WI, USA), in a final volume of 50 μL. PCR amplification was performed by using standard conditions with varying annealing temperatures (Table 1). All PCR results were analyzed on 1% Table 1. Primers used for amplication and sequencing in this study
Primer GES VEB
863
56
542
55
927
50
488
56
780
58
813
54
260
50
341
50
860
49
843
55
743
57
796
52
[15]
F: ATTTCCCGATGCAAAGCGT R: TTATTCCGGAAGTCCCTGT
PER-2
F: ATGAATGTCATCACAAAATG R: TCAATCCGGACTCACT
IMP
F: CATGGTTTGGTGGTTCTTGT R: ATAATTTGGCGGACTTTGGC
METHODS
F: ATTGGTCTATTTGACCGCGTC R: TGCTACTCAACGACTGAGCG
NDM
1. Bacterial strains and antibiotic susceptibilities
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F: ATGCGCTTCATTCACGCAC
Amplicon Tm Reference size (bp) (°C)
R: CTATTTGTCCGTGCTCAGGA
VIM
The study was performed at the Bezmialem Vakif University Hospital in Turkey between July 2012 and March 2013. During the study period, all non-duplicate clinical isolates of K. pneumoniae obtained from inpatients that exhibited a reduced sensitivity to carbapenems were characterized. Clinical isolates were identified by using standard microbiological procedures and VITEK-2 (bioMerieux, Marcy-l’Etoile, France). The identification of all isolates was confirmed by 16S rDNA sequencing [12]. Susceptibility testing was performed by using the following antibiotics: ampicillin, ampicillin/sulbactam, piperacillin, piperacillin/ tazobactam, cefuroxime, ceftazidime, cefoperazone/sulbactam, cefepime, amikacin, gentamicin, netilmicin, tobramycin, ciprofloxacin, levofloxacin, tetracycline, trimethoprim/sulfamethoxa-
5´-3´ Sequence
F: TGGAATTGCCCAATATTATGC R: TCAGCGCAGCTTGTCGGCCATGC
CTX-M1 group CTX-M2 group TEM
F: GCGTGATACCACTTCACCTC
[16]
R: TGAAGTAAGTGACCAGAATC F: TGATACCACCACGCCGCTC R: TATTGCATCAGAAACCGTGGG F: AGTATTCAACATTTYCGTGT R: TAATCAGTGAGGCACCTATCTC
SHV
F: ATGCGTTATATTCGCCTGTG R: TTAGCGTTGCCAGTGCTC
OXA-48
A: TTGGTGGCATCGATTATCGG
[7]
B: GAGCACTTCTTTTGTGATGGC KPC
A: CGTTCTTGTCTCTCATGGCC B: CCTCGCTGTGCTTGTCATCC
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agarose containing 0.5 μg/mL ethidium bromide and subsequently visualized under UV light. The PCR products for NDM, OXA-48, and TEM β-lactamases were cloned into the pGEM-T Easy vector (Promega) and then sequenced by Macrogen (Amsterdam, The Netherlands). Sequencing results were analyzed by using the alignment search tool, BLAST (http://www.ncbi. nlm.nih.gov/BLAST) [14], and the multiple sequence aligment program CLUSTALW2 (http://www.ebi.ac.uk/Tools/msa/clustalw2/). The remaining genes were evaluated according to their molecular size and PCR results of control groups which were defined as β-lactamase gene carrier bacteria in the earlier studies [7, 15, 16].
3. Repetitive Extragenic Palindronic (REP)-PCR analysis REP-PCR was used in the genotyping of K. Pneumoniae isolates. The primer pair REP 1 (5´-IIIGCGCCGICATCAGGC-3´) and REP 2 (5´-ACGTCTTATCAGGCCTAC-3´) was used [17], and the amplification reaction was performed as previously described [15]. The amplified DNA fragments were separated by electrophoresis on a 1.5% agarose gel with a 1-kb DNA ladder (Sigma Chemicals, Ontario, Canada) as the size marker. The amplified DNA bands were visualized under UV light following ethidium bromide staining, and the banding patterns of each strain were captured by using an UVP bioimaging system (UVP, Upland, CA, USA). The REP-PCR fingerprints were analyzed by using the Phoretix gel analysis package (Nonlinear USA, Inc., Durham, NC, USA).
RESULTS A total of 37 K. pneumoniae isolates were identified during the study period and investigated. All isolates were from hospitalacquired infections, with the majority from patients in the intensive care unit (n = 23), followed by patients on surgical (n = 7) and medical wards (n = 7). Of the 37 isolates, 16 (43%) were
associated with bloodstream infections. The remaining isolates came from wounds (n = 7, 19%), sputum (n = 6, 16%), tracheal aspirates (n = 6, 16%), cerebrospinal fluid (n = 1, 3%), and a catheter sample (n = 1, from anesthesia unit). According to the antibiotic susceptibility profiles, all 37 isolates were resistant to ampicillin, ampicillin/sulbactam, piperacillin, piperacillin/tazobactam, ceftazidime, cefoperazone/sulbactam, cefepime, imipenem, and meropenem. The lowest levels of resistance were observed for colistin (2.7%) and tigecycline (11%), followed by amikacin (19%) and trimethoprim/sulphamethoxazole (21.6%). Of the 37 K. pneumoniae isolates, 98% carried a carbapenemase gene. More specifically, we detected OXA-48 in 32 isolates (86%) and NDM-1 in seven isolates (19%). No other carbapenemases were identified within this group. We also observed a high prevalence of ESBLs, with all strains carrying a CTX-M-2 group β-lactamase and 23 isolates (62%) carrying CTX-M-1. SHV-1-like and TEM-1-like β-lactamase were also common (97% and 92% of strains, respectively). One of the bla TEM genes was identified as bla TEM-166, a variant of bla TEM-1b that has one amino acid change at position 53 (Arg53Gly). The combination of β-lactamases relative to the site of isolation and susceptibility test results are shown in Table 2. Importantly, all NDM-1-producing strains were isolated from blood. Furthermore, all blood isolates carried at least one carbapenemase (OXA-48 or NDM-1), and two (13%) carried both OXA-48 and NDM-1. The most common combination of β-lactamases in a given strain was OXA-48, SHV-1-like, TEM-1-like and CTX-Mtype β-lactamase, with 76% of strains observed with this combination. All 37 K. pneumoniae strains were typed by REP-PCR (Fig. 1). We observed seven predominant genotypes; the majority of strains (46%) clustered into genotype 6, while 19% and 16% of the isolates clustered in genotype 3 and 1, respectively. Of the 17 genotype 6 isolates, 11 were isolated from the anesthesia
Fig. 1. Repetitive extragenic palindronic (REP)-PCR profiles of 37 Klebsiella pneumoniae isolates. Seven different genotypes were observed, and most isolates clustered into genotypes 1, 3, and 6. http://dx.doi.org/10.3343/alm.2015.35.6.595
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Table 2. Epidemiological properties of β-lactamase-producing Klebsiella pneumoniae isolates
Isolate
Specimen
Units
Dates
Treatment
Outcome
Susceptible antibiotics
bla NDM-1 bla OXA-48 bla CTX-M1 bla CTX-M2 bla TEM
bla SHV
1
Blood
AR
Feb. 2013 SCF, CIP, TIG
Exitus
AK, TIG, CST, SXT
-
-
+
+
+
+
2
Blood
Gas
Feb. 2013 NT
Exitus
AK, CN, TIG, CST, SXT
-
+
+
+
-
+
3
Blood
Neu
Feb. 2013 TZP, SXT
Other
CN, TIG, CST, SXT
+
+
+
+
+
+
4
Blood
Neu
Feb. 2013 SXT, SCF
Other
TIG, CST, SXT
+
-
+
+
+
+
5
Blood
TS
Jan. 2013 NT
Other
AK, TIG, CST
-
+
+
+
+
+
6
Sputum
TS
Jan. 2013 NT
Other
AK, CST
-
+
+
+
+
+
7
Wound
RO
Jan. 2013 NT
Other
AK, CN, TIG, CST, SXT
-
+
+
+
-
+
8
Sputum
TS
Jan. 2013 NT
Other
AK, TIG, CST, SXT
-
+
+
+
+
+
9
Blood
IM
Jan. 2013 SCF, TIG
Other
AK, TIG, CST, SXT
-
+
+
+
+
+
10
Sputum
RICU
Jan. 2013 NT
Other
AK, CN, NET, TOB, TIG, CST, SXT
-
+
-
+
+
+
11
Sputum
RICU
Jan. 2013 SCF, TIG
Exitus
AK, CN, NET, TOB, TIG, CST, SXT
-
+
-
+
+
+
12
Wound
TS
Jan. 2013 CST, TIG
Other
AK, CST
-
+
+
+
+
+
13
Blood
PS
Jan. 2013 NT
Other
TIG, CST
+
+
+
+
+
+
14
Blood
AR
Jan. 2013 SCF, CST
Exitus
AK, CN, NET, TOB, TIG, CST, SXT
+
-
+
+
+
+
15
Tracheal aspirate
AR
Dec. 2012 SCF, TIG
Exitus
AK, TIG, CST, SXT
-
+
+
+
+
+
16
Blood
AR
Dec. 2012 TIG, SXT, CST
Exitus
AK, TIG, CST, SXT
-
+
+
+
+
+
17
Blood
Uro
Dec. 2012 NT
Other
AK, CN, TIG, CST, SXT
+
-
+
+
+
+
18
Wound
GS
Dec. 2012 MEM, TIG
Other
AK, CIP, LEV, TET, TIG, CST, SXT
-
+
+
+
-
+
19
Tracheal aspirate
AR
Dec. 2012 TIG, SUL
Treated
CN, TIG, CST, SXT
-
+
+
+
+
+
20
Blood
AR
Dec. 2012 CST, TIG
Exitus
AK, TIG, CST, SXT
-
+
+
+
+
+
21
Catheter
AR
Dec. 2012 TIG, CST
Exitus
AK, TIG, CST, SXT
-
+
+
+
+
+
22
CSF
AR
Nov. 2012 SXT, MEM, CIP
Exitus
AK, TIG, CST, SXT
-
+
+
+
+
+
23
Sputum
RICU
Feb. 2013 SCF, TIG
Exitus
AK, CN, TIG, CST, SXT
-
+
-
+
+
+
24
Blood
RICU
Oct. 2012 TIG, SUL
Exitus
TIG, SXT
-
+
-
+
+
+
25
Blood
AR
Oct. 2012 TIG, SUL
Exitus
TIG, CST, SXT
+
-
-
+
+
+
26
Wound
Gas
Nov. 2012 NT
Other
AK, TET, TIG, CST, SXT
-
+
-
+
+
-
27
Wound
AR
Jul. 2012 MEM, CIP, TIG, AK
Other
CN, CST
-
+
+
+
+
+
28
Blood
AR
Oct. 2012 SCF, CIP
Other
AK, TIG, CST, SXT
-
+
-
+
+
+
29
Wound
AR
Oct. 2012 CST, SUL, TIG, AK
Exitus
AK, TIG, CST, SXT
-
+
-
+
+
+
30
Tracheal aspirate
AR
Nov. 2012 TIG, RIF
Other
AK, TIG, CST, SXT
-
+
-
+
+
+
31
Tracheal aspirate
AR
Nov. 2012 TIG, SUL, CN, MEM, CST
Exitus
AK, TIG, CST, SXT
-
+
-
+
+
+
32
Tracheal aspirate
AR
Nov. 2012 NT
Exitus
AK, TIG, CST, SXT
-
+
-
+
+
+
33
Blood
AR
Aug. 2012 CST, CSF, TIG
Other
AK, CN, CST
-
+
-
+
+
+
34
Wound
Uro
Jul. 2012 NT
Other
AK, CN, TIG, CST
-
+
+
+
+
+
35
Sputum
AR
Nov. 2012 CST, SUL, TIG
Exitus
AK, TIG, CST, SXT
-
+
+
+
+
+
36
Blood
AR
Sep. 2012 CST, TIG
Other
AK, TIG, CST
-
+
-
+
+
+
37
Tracheal aspirate
AR
Nov. 2012 NT
Other
AK, TIG, CST, SXT
-
+
-
+
+
+
Abbreviations: AR, anesthesiology and reanimation; Gas, gastroenterology; Neu, neurology; TS, thoracic surgery; RO, radiation oncology; CP, chest diseases polyclinic; IM, internal medicine; RICU, respiratory intensive care unit; PS, plastic surgery; Uro, urology; GS, general surgery; TZP, piperacillin/tazobactam; SCF, cefaperazone/sulbactam; AK, amikacin; CN, gentamicin; NET, netilmicin; TOB, tobramycin; CIP, ciprofloxacin; LEV, levofloxacin; TET, tetracycline; TIG, tigecycline; CST, colistin; SXT, trimethoprim/sulfamethoxazole; MEM, meropenem; SUL, sulbactam; RIF, rifampicin; CSF, cerebrospinal fluid; NT, Not treated; Other, the patient was transferred into a different service.
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Iraz M, et al. β-Lactamase genes in carbapenem-resistant K. pneumoniae
and reanimation intensive care unit (ICU), and three were isolated from the thoracic surgery unit. Of the seven genotype 3 isolates, two were isolated from patients who were in the neurology ward at the same time, and two were isolated from the anesthesia and ICU. The remaining isolates were distributed among other wards in the hospital over different times during the 9-month study period (Table 2).
DISCUSSION K. pneumoniae is an opportunistic pathogen that causes community- and hospital-acquired infections, such as bloodstream and urinary tract infections, and pneumonia [18]. Currently, carbapenems are the most potent antimicrobial agents used in the treatment of serious infections caused by multidrug-resistant gram-negative bacteria. However, carbapenem resistance in Enterobacteriaceae is emerging in Turkey [8, 9] and in other parts of the world [5]. Here, we described the distribution of major β-lactamases in a contemporary group of extensively drug-resistant K. pneumoniae isolates from Turkey. We observed that 19% of isolates carried the NDM-1 carbapenemase, and that all of these isolates came from bloodstream infections and from patients with no recent travel history, suggesting local acquisition. OXA-48 carbapenemases were highly prevalent, and were occasionally found co-existing with NDM-1. Compounding these data was that over 2.7% of isolates were resistant to colistin, a finding that is novel for Turkey and one with serious therapeutic implications. The majority of isolates came from patients in the ICU. BLs have been reported worldwide, and their genes are often located on plasmids and integrons [19]. NDM is a novel MBL enzyme, and its variants are the latest carbapenemases to be recognized and reported worldwide. NDM-1 was first identified in a carbapenem-resistant K. pneumoniae strain from the urine sample of a Swedish patient of Indian origin who traveled to New Delhi [20]. Since then, more than 40 countries have reported NDM-producing isolates [21, 22]. NDM enzymes have predominately been identified in Enterobacteriaceae , but have also been described in non-fermenters and in members of the Vibrionaceae family [23]. Occasional community-acquired NDM-1-positive isolates have been reported [24], however, the overwhelming majority of isolates have a nosocomial origin. In our study, all isolates were from nosocomial infections and all were from seriously ill patients. All of the NDM-1-positive isolates (n = 7) were recovered from blood samples, and six of these were from patients in the ICU. All pahttp://dx.doi.org/10.3343/alm.2015.35.6.595
tients were uninfected at admission, and the organisms were isolated from patients 48 hr or longer after admission. In Turkey, NDM-1 was first reported in 2011 in a K. pneumoniae isolate from a leukemia patient originally from Iraq [19]. Later, another team investigated NDM-1 in various carbapenemase-resistant gram-negative bacilli, but with negative results [25]. To our knowledge, the current study is the largest report demonstrating the presence of NDM-1 in K. pneumoniae in Turkey. Our results indicate that seven out of 37 (19%) carbapenem-resistant K. pneumonia isolates were positive for NDM-1, an alarming result as efforts to identify NDM-1-producing isolates in 2011 showed negative results. Another β-lactamase type known as OXA-48 was initially identified in Turkey from a K. pneumoniae isolate from Istanbul, and thereafter, outbreaks of OXA-48-producing K. pneumoniae have been described in Turkey [8-10]. To date, 11 variants of OXA-48 have been described [26]. Recently, the molecular characterization of carbapenem-resistant K. pneumoniae in a tertiary university hospital in Turkey was performed on 94 isolates. OXA-48 and NDM-1 were produced in 91.5% and 4.3% of isolates, respectively [27]. In our study, OXA-48 and NDM-1 were observed in 86% and 19% of isolates, respectively. This study reveals that the NDM-1 resistance gene is increasing in prevalence in Turkey. This increase poses a significant threat to Turkish infection control efforts. TEM or SHV β-lactamase derivatives have been the most prevalent ESBLs in nosocomial pathogens since the 1980s. However, starting from 1995, CTX-M-type ESBLs have emerged worldwide, including Europe, Asia, South America, and Canada [13, 28]. We demonstrated that these CTX-M-type ESBLs are also highly prevalent in multidrug-resistant K. pneumoniae in Turkey. In a multicenter study conducted in Turkish hospitals, PCR analysis was used for ESBL typing, and CTX-M was the most prevalent enzyme type among blood isolates (71%) of Escherichia coli and K. pneumonia , followed by TEM (49%) and SHV (47%) type enzymes [29]. The CTX-M family can be classified into five major groups: CTX-M-1, CTX-M-2, CTX-M-8, CTX-M-9, and CTX-M-25. CTXM-15, which belongs to the CTX-M-1 group, is the most widespread of these enzymes around the world [30]. Ours is the first report of CTX-M-1 and CTX-M-2 in ESBL-producing carbapenem-resistant K. pneumoniae strains from Turkey. CTX-M-2 was the most prevalent β-lactamase, found in 100% of isolates, followed by SHV (97%), TEM (92%), and CTX-M-1 (62%). The continued monitoring of antimicrobial resistance to β-lactams in each region of Turkey is urgently required to assist with the de-
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velopment of control strategies for hospital infections. In conclusion, our study of carbapenem-resistant, K. pneumoniae isolates identified the largest number of NDM-1-producing strains reported from Turkey, and revealed that these strains were highly resistant to a range of clinically important antibiotics. Furthermore, these isolates often carried the OXA-48 carbapenemase, and were associated with serious infections of the bloodstream. All strains also carried multiple ESBLs, with bla CTX-M-2 being the most common. The recent emergence of NDM-1-producing K. pneumoniae on the background of the highly prevalent OXA-48 and CTX-M type ESBLs is alarming for our country.
Acknowledgments This work was supported by Recep Tayyip Erdoğan University Research Fund Grants BAP-2013.102.03.12 and BAP-2013. 102.03.13. A.Y.P was supported by an Australian National Health and Medical Research Council Career Development Fellowship (APP1047916).
Authors’ Disclosures of Potential Conflicts of Interest No potential conflicts of interest relevant to this article were reported.
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